X-linked agammaglobulinemia (XLA) results from deficient function of Bruton's tyrosine kinase (Btk) and is characterized by a severe block in early B-cell development. B cells that express wildtype Btk exhibit a strong selective advantage in vivo; suggesting that introduction of a normal Btk cDNA into autologous hematopoietic stem cells (HSC) may lead to long-term immunologic reconstitution in XLA. In previous work, we demonstrated that both gamma-retroviral and B lineage-specific, lentiviral (LV) Btk gene therapy can lead to rescue of Btk-dependent, B lineage development and function in vivo. Notably, in addition to its role in B cells, Btk is activated via multiple receptors expressed on myeloid cells implying a broader role for Btk in host immune responses. To optimize the expression profile of the Btk transgene in both B and myeloid lineage cells, we developed a novel LV platform using the endogenous Btk promoter (Btkp) in association with two alternative elements [the IgH chain enhancer Em or a ubiquitous chromatin opening element (UCOE) element]. Using these LV to express codon-optimized human Btk, we observed rescue of Btk-dependent, B-lineage development and function; and restoration of Toll-like receptor (TLR) signaling in myeloid cells. Together, these data strongly support our goal of using a Btkp-based LV in a future gene therapy trial for patients with XLA. Several key issues; however, remain to be addressed before this approach can be moved forward into patients. First, we need a comprehensive in vivo comparison of the safety and efficacy of EmBtkp vs. UCOE.Btkp LV in both murine XLA and primary stem cells derived from XLA patients. Second, we need a detailed assessment of potential genotoxicity of these candidate LV. Accordingly, this proposal is designed to test the hypotheses that: (1) LV utilizing the endogenous Btk promoter will mediate sustained, temporally appropriate levels of Btk gene expression and rescue of B and myeloid function in murine and human cells; (2) Efficient Btk rescue will also be achieved using a non-myeloablative marrow conditioning regimen that closely mimics a future clinical approach in XLA; and (3)These LVs will exhibit little or no risk of vector- mediated mutagenesis. PUBLIC HEALTH RELEVANCE: X-linked agammaglobulinemia (XLA) results from deficient function of Bruton's tyrosine kinase (Btk) and is characterized by a severe block in early B-cell development. Development of genetic therapy for X-linked agammaglobulinemia is an important next step in the evolution of gene therapy for primary immunodeficiency disorders. Our laboratory has extensive expertise in both functional and gene transfer studies of Btk and has pioneered studies using both gamma-retroviral and lentiviral vectors expressing human Btk for rescue of B cell function in a murine XLA model. This application is designed to address the remaining questions required to move forward to a clinical trial. Our work will identify an optimal lentiviral vector capable of rescuing Btk function in primary B and myeloid cells from XLA mice and humans. In addition, we will extensively test candidate vectors for relative safety using several distinct assays. Because B cell function impacts a broad array of human diseases, studies in this rare disorder is also likely to benefit future therapies for more common immune disorders.